Project Summary/Abstract This proposal brings together four research teams with expertise in cancer genome sequencing, single-cell sequencing, and cancer cell biology to advance single-cell sequencing technology to tackle compelling questions in basic and translational cancer research. It responds to RFA-CA-17-023, Cancer Moonshot Initiative,andproposestocreateandvalidateaplatformofexperimentalandanalyticalapproachesforprecise single-cell sequencing of human samples based on the most recent advance in single-cell whole-genome amplification, Linear Amplification after Transposon Insertion (LIANTI), developed by the Xie laboratory. The immediate goal is to further develop LIANTI and augment bioinformatic analysis for single-cell LIANTI sequencing.Aim1focusesondevelopingahighthroughputversionofLIANTI.Aim2focusesondeveloping computational methods that utilize the digital and linear nature of LIANTI sequencing data. Aim 3 describes strategiesandexperimentalmodelsthatwillbeusedtovalidateandbenchmarksingle-cellLIANTIsequencing performanceanddemonstrateitsapplicabilitytotumoranalysis.Overall,ourgoalistooptimizeandimplement the most precise single-cell sequencing technology for variant detection in single cancer or somatic cells. Successoftheprojectwillresultinanintegratedplatformthatovercomesprevailingimpedimentsinsingle-cell analysis and can have a transformative impact in multiple areas of cancer research, including tumor genetic heterogeneity, circulating tumor cells, primary/metastasis relationship, drug resistance, and benign-to- malignancytransformation.